Project Summary Overexpression of hTERT is a hallmark of cancer and is associated with oncogenesis, tumor maintenance, and resistance to conventional chemotherapy. Elongated telomeres at the ends of chromosomes are a hallmark for cancer in the early stage of tumorigenesis and are associated with genetic instability. As a consequence, suppressed telomerase in normal cells is highly activated to immortalize tumor cells. There are several strategies to overcome activated telomerase for potential therapeutic treatment, including targeting the telomeric G-quadruplex with small molecules, but other strategies exist, which include targeting hTERT and hTR via gene therapy. However, as yet no strategy for inhibition of telomerase has been successfully translated into a clinically useful agent. Agents that target the telomeric G-quadruplex mediate their effects by erosion of telomere length, which can take several weeks or even months. This is a distinct disadvantage for cancer therapy. On the other hand, the telomerase-independent effects occur during a much more therapeutically relevant timeframe, and this is the focus of this proposal. Several groups have demonstrated recently that many different kinds of tumors have somatic mutations in the hTERT promoter region ?124, ?124/125, ?138/139, and ?146 bases from the ATG start site. As a possible explanation for the enhanced transcription of hTERT, the C/T mutation in the sense strand (or G/A in the antisense strand) is proposed to generate an ETS element (CCTT/GGAA) to increase binding of the ETS transcription factor for activation of hTERT transcription. More significantly, these mutations are also located in the G-quadruplex with the 3:26:1 loop configuration or in the corresponding C-quadruplex-forming region. We have shown that mutations in the loop lead to misfolding of the G-quadruplex that in turn leads to loss of hTERT silencer activity. Eighty one percent of adult GBM patients have ?124 and ?146 mutants which are associated with the cooperative folding process and lead to misfolding of the silencer element. Annually in the United States, glioblastoma multiforme (GBM) afflicts nearly 15,000 new patients. We have identified a small drug-like pharmacological chaperone (pharmacoperone) molecule that acts at an early step in the cooperative folding pathway to redirect the G-quadruplex misfolding, which reinstates the correct folding pathway and reduces hTERT activity. This compound, TG-4260, directly decreases the transcription activity of the WT and the ?124, ?124/125, ?138/139, and ?146 mutants to a similar extent and suppresses the downstream gene BCL2, which activates caspase-3 and produces cell-cycle arrest, leading to cell death. Finally, TG-4260 significantly inhibits telomerase and shortens telomere length after five days of treatment and induces a senescence-like phenotype. The restoration of the silencer function of the WT G- quadruplex silencer element by drugs of this type has important applications in treating patients with cancers such as GBM, where a significant number of the patient population carry these somatic mutations.